In the age of short read sequencing you could treat the cells with very harsh physical disruption as there was little chance that your DNA extraction could shear your DNA to shorter fragments than the machines could sequence. However, with the emerging long read sequencers short fragments are suddenly no fun and people have started […]
Since November we have run 11 PromethION flow cells, generating >900 Gbp of data. It has been a journey of highs and lows, so we want to share what we have learned along the way. Our first lesson: it is important to use those flow cells ASAP! Despite our lab’s earlier success with a three […]
After unboxing the Nanopore alpha-beta PromethION device some weeks ago and having our IT support/linux ninja help us integrate yet another box into the university network, we finally got around to run some PromethION sequencing again. We updated the PromethION software to Minknow 2 inserted a flowcell and it passed QC with 7000+ pores available […]
Each year, the Roblon foundation awards the Roblon Prize of 100,000 DKK (~16,500 USD), to a master thesis from Aalborg University acclaimed to fulfill the following: “The thesis should be of highest quality and innovative. It should have a positive effect on the research field as well as the future research and career of the […]
Day 2 at the NCM17 started, again, with Oxford Nanopore CEO Gordon Sanghera taking the stage, once again stating his dream that sequencing should be available to anybody, anytime, anywhere. There are currently around 12,500 mainframe DNA sequencers around the globe, a number he believes will be passed by ONT in a not too distant future. […]
All the gear We start out with blazing punk rock (things are never too boring at Oxford Nanopore meetings) and plenty of promises from Oxford Nanopore CEO Gordon Sanghera. He gave us updates on various products, such as PromethION flow cells, which have been harder to produce than expected. However, we and others have now sequenced […]
Oxford Nanopore is hosting their annual community meeting in New York City this week. I’ll be there, thanks to being in top 5 of their one more flowcell competition – thanks to all that voted! Although not as “big” as the London Calling conference they host, the line-up of speakers is impressive and promises talks […]
Hi everybody and welcome to my first blog post at Albertsen Lab. As a newly started PhD student, I have engaged myself with the simple, yet Herculean task of populating the tree-of-life. As most people are aware of, microorganisms are more or less inescapably present in all places of the world — no matter how […]
My name is Peter Rendbæk, and I’m currently a master student in the Albertsen lab. The overarching aim of my master project, is as a pre-test for several of the new big projects in the group, which focus on applying the on-line bacterial identification for process control at wastewater treatment plants. Hence, last couple of months I have been working on the project “Developing methods for on-site DNA sequencing using the Oxford Nanopore MinION platform”. The MinION has improved a lot since its release three years ago, and it can now be used to make rapid determination of bacterial compositions.
The potential for this fast and mobile DNA-sequencing is mind-blowing. However, given that the technology is here now (!), there has been relatively little focus on portable, fast, easy and robust DNA extraction. Hence, I’ve spent the last months on trying to develop a fast, cheap, mobile, robust and easy to use DNA extraction method.
There is a significant amount of bias connected with DNA extraction, but the bias associated with wastewater treatment samples has been investigated in depth. However, the “optimized method” is not suited for on-site DNA-extraction. There are 3 principle steps in DNA extraction, cell lysis, debris removal and DNA isolation, which I will cover below and discuss how I simplified each step.
In general, complex samples require bead beating for cell lysis and homogenization. The problem is that our in-house bead-beating is done by a big table top tool weighing 17 kg, which makes it hard to transport. However, I came across a blog post from loman labs about sample preparation and DNA extraction in the field for Nanopore sequencing. In the blog post, the possibilities of a portable bead beater outlined, by the use of a remodeled power-tool. I thought this was interesting, so I went out and bought an Oscillating Multi-Tool cutter and tried this with lots of duct tape…
The amazing part was that it worked! But the problem was that the samples would get “beaten” differently depend on how you taped the sample to the power-tool, which could give rise to variation large variations in the observed microbial community.
I solved this by 3D printing an adapter to the power-tool that fits the bead-beater tube (Finally, a good excuse to use a 3D printer!). I used Solidworks to design the adapter and collaborated with our local department of mechanical and manufacturing engineering (m-tech) in 3D printing it. You can make your own by simply downloading my design from Thingiverse (It did take a few iterations to make it durable enough, and I still use a little duct tape..).
After the bead beating, the cell debris removal is done by centrifugation. Our “standard” protocol recommends centrifugation at 14000 x G for 10 minutes at 4 C. However, in our minds that seemed a little extensive and requires a huge non-transportable centrifuge… Alternatively, there are a lot of possibilities to use small, easy to transport and easy to use centrifuges if we do not have to centrifuge at 14.000 xG at 4 C. There is even the possibility to 3D print a hand-powered centrifuge. However, I did not follow this path, as it seems a bit dangerous… After several tests, we discovered that a simple table top centrifuge could do the job perfectly well, using 2000 xG for 1 min at room temperature if we combined it with the DNA isolation described below.
The last step is DNA isolation, I tried several different methods, but we got the idea to simply use Agencourt AMPure XP that is routinely used in e.g. PCR purification (we 10 diluted the AMPure XP beads 1:10 to save some money and it seems to work just as good). And… It works..
So, now you have an overview of the method I developed. The most amazing part is that it works! It takes 10-15 minutes from the sample is taken until you’ve got ready DNA for use, compared to 60+ minutes for our “standard” protocol. Furthermore, it requires inexpensive equipment that can be carried in a small suitcase. So, just to prove that this approach is fast, I filmed myself doing the DNA extraction with a GO-PRO camera, as you can see below.
The next part is to test the MinION in the lab. How, fast can we identify bacteria and is the extracted DNA compatible with the downstream library preparation, which we hope to do on the our new and shiny Voltrax (which is now moving liquids!).